We have found that cytochrome c1 in beef heart mitochondria behaves as two distinct thermodynamic species. One has an Em 200 mV and a spectral peak at 555 mm and the other has an Em 250 mV and a peak at 553 nm. Both exhibit n values of 2. Current views are that cytochrome c1 is a single thermodynamic entity with Em 230 mV, a peak at 553 nm, and an n value of 1. Our ability to resolve the two species is based on our unique procedures which collect and analyze much more data than is normally used. Our findings are confirmed by two independent analysis. Using a limited amount of data and the older analytical procedure yields the traditional findings for a single species. A theoretical analysis of Q-cycle mechanisms in respiration, in terms of thermodynamics and kinetics, has been completed. Initial studies with mitochondrial and purified cytochrome aa3 indicate the presence of three thermodynamic species with Em values of 190 mV, 250 mV and 340 mV and n values of 2, 2, and 1, respectively. Two new experimental systems were developed. One performs computer-controlled Coulometry while collecting complete optical spectra and the other performs potentiometric titrations using working voltages controlled by a potentiostat.